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Team:Virginia/Notebook
From 2013.igem.org
Notebook
June
July
August
September
- After much back and forth, the 2013 Virginia iGEM team has chosen a project! We will be researching the formation of minicells via the expression of the FtsZ gene! The protocols have been researched and we have begun our experiments!
- (photo: Elli Williams)
- Our first protocol in lab was to extract the bacterial genome on June 17th. The next day we isolated FtsZ using a PCR reaction with primers designed by our team. This isolation was confirmed via gel electrophoresis; FtsZ is about 1200bp and the gel confirmed isolation and extraction! The team’s first attempt at ligating our isolated FtsZ onto the iGEM chloramphenicol backbone was on June 19th. Unfortunately, days later it was confirmed it did not work. After addressing issues with antibiotics and media, we finally achieve transformation and selection success! Colonies were selected and grown up in liquid LB + Chloramphenicol 25mg/L. These colonies were miniprepped on June 29th, and the plasmid was extracted. The DNA was digested to confirm the FtsZ ligated to the backbone. After confirmation of the ligation, the plasmid was sent to sequencing to confirm absolutely the FtsZ is intact.
July
- The team drew out of compound Biobrick formation plan.
- On July 9th, the team ligated FtsZ with the ribosome binding site of choice (BBa_B0034) as well as GFP (BBa_b0015) to the same ribosome binding site. We received and analyzed the data from sequencing. Although it appeared incorrect at first, we have officially isolated the FtsZ gene and it is present in our Biobrick! Unfortunately, on July 12th a gel confirmation indicated that the ligation failed. A strain of minicells from the University of Texas Health Science Center at Houston was received on July 18th. This was a strain of minicells derived from the silencing of minCDE gene. These will form a good basis for comparison to our minicells.
- After many failed transformations and ligations, members of the team sat down with the advisors to discuss and resolve these problems. New protocols were formed and the outlook is bright! A new restriction digest was performed with the ribosome binding site, GFP and our FtsZ. These DNA samples were isolated and ligated together according to the compound Biobrick formation plan using the new protocols. On July 21st, the team used the new transformation protocol to transform the ligated material. This time the transformation was a success and the colonies were grown up! The successful transformants were miniprepped. The team ran a confirmatory digest and gel on the DNA and it was sent off for sequencing on the 24th. An IPTG inducible promoter (BBa_R0011) was ligated onto the ribosome binding site + FtsZ construct. A terminator (BBa_B0015) was ligated onto the ribosome binding site + GFP. This was confirmed by another digest and gel. These samples were also sequenced.
- So finally, after a month of trial and error, the team has found their stride with a winning combination of good protocols and good results! The team also received a minicell strain for Yale that utilizes Ftsz instead of minCDE.
August
- In August our team hit a major snag. Our isolated FtsZ has an illegal restriction site and was most likely the cause of our problems in the beginning. The gene has been ordered with a mutation in the restriction site. In the meanwhile, the team attempted to utilize site directed mutagenesis. Although Biobrick formation was at a roadblock, the team continued with purification and applications. The sucrose gradient minicell purification protocol was competed using the WM1032 strain from Yale. The Promoter+RBS construct (BBa_K215000) was received from iGEM. The AiL plasmid for our applications team was received and inoculated.
- On August 13th the Ribosome Binding Site + GFP + Terminator and the Ribosome Binding Site + GFP was sent off for sequencing. The team also received our long awaited for synthesized FtsZ gene without the illegal cut site. All previous “BioBricks” completed in August were redone with the correct FtsZ and confirmed with a digest and gel.
September
- HA tags were prepared for further characterization of our FtsZ Biobrick. The team combined Promoter•RBS•FtsZ with our terminator as well as Promoter•RBS•FtsZ with RBS•GFP•Terminator. These are our final constructs that we will send in to iGEM. Unfortunately, the Promoter+RBS+FtsZ+Terminator was not a successful ligation, but the later construct was successful. This DNA was sent in for sequencing and sent off to iGEM on the 10th. For BioBrick submission, the team made the following pages on 9/12/13:
- BBa_K1011000 – FtsZ on psB1C3
- BBa_S05211 – BBa_B0034 (RBS) + BBa_E0040 (GFP)
- BBa_S05212 – BBa_S05211 + B0015
- BBa_S05213 – BBa_K215000 + BBa_K1011000
- BBa_K1011001 – Promoter•RBS•FtsZ•RBS•GFP•Terminator
